Project Summary: The mammalian brain is an exquisite structure, with sheer metrics of complexity that surpass by orders of magnitude any other observed in nature. And yet, the ?nite amount of information stored in the genome, 7.5 gigabytes in humans ?the size of a thumb drive, su?ces to hold the blueprints to construct the circuits that create the brain. The question arises, through what cell biological mechanisms is genetic information decompressed and elaborated into the elements of brain circuitry? The cerebral cortex in particular exempli?es this problem, with extreme hyperconnectivity, multitude of long-range projections, and molecular heterogeneity of the highest order. These features endow the cortex with its remarkable functions, however they also pose a scienti?c challenge to investigation. We propose to tackle the question of how cortical circuits develop by taking an unconventional approach, one that experimentally embraces ?rather than circumvents? these outstanding features, and does so in a quantitative, high-throughput manner. We aim to achieve this using a new set of innovative in vivo molecular approaches that we have developed to directly target and manipulate circuits in the developing cerebral cortex. Having recently identi?ed the subcellular proteomes of a growing cortical projection, we propose to target these molecules in their native circuit elements using in utero CRISPR genome editing to investigate their function in circuit development in vivo. We additionally propose to use optical controls of proteins delivered in utero, to selectively activate and inactivate molecular functions with light at subcellular foci of circuit development. We combine these with high-throughput platforms of modular DNA assembly, whole-brain phenotype screening, and an innovative quanti?cation approach that increases sensitivity, rigor, and data integration. Using these work?ows, we have already ascribed new circuit development functions to a number of genes, and aim to do so across the local proteome of cortical connectivity in the mouse. Success in bridging the gap in our understanding between genes and the formation of brain circuitry will be transformative in uncovering how genetics underly circuit pathologies in neurological and psychiatric disease, and contribute towards understanding how the one of the most remarkable structures of nature is formed.